Distant metastases cause more than 90% of cancer-related mortality. Children suffering from osteosarcoma, where the typical route of spread is from bone to lung, are no exception. Despite the development of numerous treatment modalities targeting the primary tumor, 40% of patients still die from metastatic progression. There is therefore a pressing clinical need to determine the factors responsible for lung metastasis in osteosarcoma to facilitate development of novel anti- metastatic therapies and prognostic biomarkers. This proposal tests the central hypothesis that epigenetic alterations at distal enhancer loci underlie metastatic competence in osteosarcoma. Based on the fundamental principle that gene enhancer elements determine cellular identity and are key in mediating cellular responses to exogenous stimuli, we mapped enhancer elements genome wide through ChIP-seq of enhancer-histone marks. Compared to non-metastatic progenitors, metastatic cells show somatic acquisition and loss of enhancer- histone marks at thousands of loci across the epigenome. We call these Metastatic Variant Enhancer Loci, or Met-VELs. Using a lung explant mouse model of pulmonary metastasis, we show that gained and lost Met-VELs converge on prometastatic transcriptional programs that are uniquely activated or repressed as the metastatic cells colonize the lung microenvironment. The findings suggest that an osteosarcoma cell's metastatic success depends on its capacity to dynamically activate and repress pro- and anti-metastatic genes in the lung microenvironment, and that this ability is encoded in the epigenome at the level of gene enhancer elements. Aim 1 tests the hypothesis that Met-VELs are ubiquitous and predictive of metastatic transcription in osteosarcoma cell lines and in patient samples. Aim 2 tests the hypothesis that Met-VEL gene activation and repression is driven by dynamic changes in the epigenetic state of enhancers and promoters, dictated by the tumor microenvironment. Aim 3 tests the hypothesis that Met-VEL clusters define metastasis-dependency genes. Successful completion of these aims will provide conclusive evidence that osteosarcoma cells hijack enhancers to metastasize to the lungs, and could reveal commonalities among metastatic cells exploitable as therapeutic targets and prognostic biomarkers in human patients.